V23: the histone code

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1 V23: the histone code The DNA of eukaryotic organisms is packaged into chromatin, whose basic repeating unit is the nucleosome. A nucleosome is formed by wrapping 147 base pairs of DNA twice around an octamer of four core histones, H2A, H2B, H3 and H4 (2 copies of each one). X-ray structure of the nucleosome core particle consisting of core histones, and DNA. Top view. Side view shows two windings of DNA and two histone layers 1

2 Post-translational modifications of histone tails The disordered histone tails comprise 25-30% of the histone mass. PNAS 1964;51:786 First report on PTMs of histones They extend from the compact histone multimer to provide a platform for various posttranslational modifications (PTMs). These modifications affect the histones' ability to bind DNA and to other histones. This, in turn affects gene expression. Strahl BD and Allis CD, Nature 403:

3 Mode of action of histone PTMs Histone PTMs exert their effects via two main mechanisms. (1) PTMs directly influence the overall structure of chromatin, either over short or long distances. (2) PTMs regulate (either positively or negatively) the binding of effector molecules. Bannister, Kouzarides, Cell Res. (2011) 21:

4 PTMs of histone tails Histone acetylation and phosphorylation effectively reduce the positive charge of histones. This potentially disrupts electrostatic interactions between histones and DNA. This presumably leads to a less compact chromatin structure, thereby facilitating DNA access by protein machineries such as those involved in transcription. Histone methylation mainly occurs on the side chains of lysines and arginines. Unlike acetylation and phosphorylation, however, histone methylation does not alter the charge of the histone protein. Bannister, Kouzarides, Cell Res. (2011) 21: By Ybs.Umich - Own work, CC BY-SA 3.0, 4

5 H4 tail : conformational dynamics All histone tails can influence chromatin compaction and accessibility, depending on - salt concentration, - construction of the nucleosome arrays, and - the type of assembly process. The H4 tail probably plays the most important role in inter-nucleosome interaction. Its middle part, the K 16 RHRK 20 segment forms a positively charged basic patch. On the H2A-H2B dimer, the glutamic acid and aspartic acid residues H2A E56, E61, E64, D90, E91, E92, and H2B E102 and E110 build up a negatively charged area, called the acidic patch. Due to the spatial proximity and the electrostatic attraction, stable salt bridges can be formed between these two parts from neighboring nucleosomes 5

6 Molecular dynamics simulations of H4-H2A/H2B-DNA system Left: Structure of two nucleosomes from crystal packing Right: the model structure used in atomistic MD simulations. Water not shown. (Green) DNA; (yellow) H3; (gray) H4; (pink) H2A; and (blue) H2B. 6

7 Acetylation effects Distribution of the distance between the H4 tail and the neighboring H2A-H2B dimer in the MD simulations. The center of mass of the backbone atoms of H4 tail residues 7 17 and H2A-H2B dimer are used for distance measurement. The middle part of the AC H4 tail is generally further away from the adjacent H2A-H2B dimer. The major population of WT is located at nm, indicating close contact between H4 tail middle part and the neighboring H2A-H2B dimer. The distribution of AC is broader, ranging from 2.2 to 3.1 nm, and the multiple peaks refer to diverse conformation clusters. The center of the major peak of the AC population (AC-3 and AC-6) is shifted 0.2 nm to the right of the WT center. 7

8 The H4 tail is basically disordered due to active electrostatic interaction with outside partners. Acetylation effects Only some low-frequency helix structures (formed by i+3 i hydrogen bonds) were found in the WT system. In the AC system, the occupancy of helix structures is twice as high. Acetylation disrupts the interaction between the H4 tail and the acidic patch. This gives the H4 tail the flexibility to form intratail hydrogen bonds. The increasing intratail interaction helps to stabilize these structures. 8

9 Protein domains bind to modified histones H3K4me3 a mark associated with active transcription is recognized by a PHD finger within the ING family of proteins (ING1-5). The ING proteins in turn recruit additional chromatin modifiers such as HATs and HDACs. Examples of proteins with domains that specifically bind to modified histones. There are more domain types recognizing lysine methylation than any other PTM. Bannister, Kouzarides Cell Res. (2011) 21:

10 Histone modification crosstalk Histone PTMs can positively or negatively affect other PTMs. The large number of histone PTMs enables tight control of chromatin structure. An extra level of complexity exists due to cross-talk between different modifications, which presumably helps to fine-tune the overall control. A positive effect is indicated by an arrowhead and a negative effect is indicated by a flat head Bannister, Kouzarides Cell Res. (2011) 21:

11 Euchromatin vs. Heterochromatin structure Eukaryotic genomes can be divided into two geographically distinct environments. (1) a relatively relaxed environment, containing most of the active genes and undergoing cyclical changes during the cell cycle. These 'open' regions are referred to as euchromatin. (2) other genomic regions, such as centromeres and telomeres, are relatively compact structures containing mostly inactive genes. These more 'compact' regions are referred to as heterochromatin. Both heterochromatin and euchromatin are enriched, and indeed also depleted, of certain characteristic histone PTMs. However, there appears to be no simple rules governing the localization of PTMs. There is a high degree of overlap between different chromatin regions. Nevertheless, there are regions of demarcation between heterochromatin and euchromatin. These 'boundary elements' are bound by specific factors such as the insulator CTCF. Bannister, Kouzarides Cell Res. (2011) 21:

12 Euchromatin Interplay of factors at an active gene in yeast. (Left) The scset1 H3K4 methyltransferase binds to serine5 phosphorylated C-terminal domain (CTD) of RNAPII, the initiating form of polymerase situated at the TSS. (Right) In contrast, the scset2 H3K36 methyltransferase binds to serine 2 phosphorrylated CTD of RNAPII, the transcriptional elongating form of polymerase. Thus, the two enzymes are recruited to genes via interactions with distinct forms of RNAPII the location of the different forms of RNAPII define where the PTMs are placed Bannister, Kouzarides Cell Res. (2011) 21:

13 Epifactors database The database EpiFactors stores detailed and curated information about 815 proteins and 69 complexes involved in epigenetic regulation. MSc thesis topic! Side view shows two windings of DNA and two histone layers Database (Oxford). 2015; 2015: bav

14 Frequency of main annotation terms of epifactor proteins Function Count Modification Count DNA modification 22 DNA methylation 7 RNA modification 30 DNA demethylation 12 Chromatin remodeling 101 DNA hydroxymethylation 5 Chromatin remodeling cofactor 41 RNA degradation 9 Histone chaperone 26 mrna editing 10 Histone modification 15 Histone methylation 127 Histone modification cofactor 12 Histone acetylation 139 Histone modification read 90 Histone phosphorylation 55 Histone modification write 158 Histone ubiquitination 61 Histone modification write cofactor 95 Histone sumoylation 2 Histone modification erase 66 Histone citrullination 4 Histone modification erase cofactor 58 TF activator 18 Polycomb group (PcG) protein 29 TF repressor 27 Scaffold protein 12 TF 53 Database (Oxford). 2015; 2015: bav

15 Most frequently occurring Pfam domains Database (Oxford). 2015; 2015: bav

16 ENCODE The ENCODE (Encyclopedia of DNA Elements) Consortium is an international collaboration of research groups funded by the National Human Genome Research Institute (NHGRI). The goal of ENCODE is to build a comprehensive parts list of functional elements in the human genome, including elements that act at the protein and RNA levels, and regulatory elements that control cells and circumstances in which a gene is active. ENCODE consortium Nature 489, 57 (2012) 16

17 ENCODE: gene expression TF binding sites Correlative models between TF binding and RNA production in K562 cells. (Left) output of the correlation models (x axis) compared to observed values (y axis). (Right) The bar graphs show the most important TFs. ENCODE consortium Nature 489, 57 (2012) 17

18 ENCODE: gene expression histone marks Correlative models between histone marks and RNA production in K562 cells. ENCODE consortium Nature 489, 57 (2012) 18

19 ChromHMM - ChromHMM is a software based on a multivariate Hidden Markov Model for learning and characterizing chromatin states. - Input data can be multiple chromatin datasets such as ChIP-seq data of various histone modifications. - The trained ChromHMM model can be used to systematically annotate a genome in one or more cell types. Manolis Kellis MIT Ernst, Kellis, Nature Methods 9, 215 (2012) 19

20 ChromHMM Example of chromatin-state annotation tracks produced from ChromHMM and visualized in the UCSC genome browser. Shown as example is the NFKB1 (subunit of nuclear factor kappa B, this TF controls more than 200 genes). Active promoter, transcription transcription + elongation, insulator before next gene MANBA Ernst, Kellis, Nature Methods 9, 215 (2012) 20

21 ChromHMM (left) which PTMs contribute to which states. (right) Relative percentage of the genome represented by each chromatin state. TSS, transcription start site; TES, transcript end site; GM12878 is a lymphoblastoid cell line. Ernst, Kellis, Nature Methods 9, 215 (2012) 21

22 Relate histone modifications to expression (i) Is there a quantitative relationship between histone modifications levels and transcription? (ii) Are there histone modifications that are more important than others to predict transcript levels? (iii) Are there different requirements for different promoter types? (iv) Are the relationships general? Martin Vingron MPI Berlin The numbers of tags for each histone modification or variant, found in a region of 4,001 base pairs surrounding the transcription start sites of 14,802 RefSeq genes, was used as an estimation of the level of histone modifications. Karlic et al., PNAS 107, 2926 (2010) 22

23 Relate histone modifications to expression Models are formulated as equations that linearly relate the levels of histone modifications to the measured expression value. N i : transformed levels of histone modification i N i = log(n i + α i ) (vector of length L) N i : number of tags in each promoter y : expression values (vector of length L). In the one-modification models, i can be any of the 39 modifications or two control IgG antibodies. In the two-modifications models, i and j cover all combinations of two modifications without repetition. In the three-modifications models, i, j, and k cover all combinations of three modifications without repetition. The full model incorporates all 41 variables. Karlic et al., PNAS 107, 2926 (2010) 23

24 Linear model for expression Predicted expression values in CD4+ T-cells using the full linear model on the x axis and the measured expression values in CD4+ T-cells on the y axis. The shades of blue indicate the density of points; the darker color, the more points. Red line : linear fit between predicted and measured expression (y = 0.99x ), which are highly correlated (r = 0.77) a quantitative relationship exists between levels of histone modifications at the promoter and gene expression levels (see slide 18 from ENCODE project) Karlic et al., PNAS 107, 2926 (2010) 24

25 Linear model for expression Comparison of prediction accuracy between all possible one-modification, two-modifications, threemodifications models, and the full model for CD4+ T- cells. Models are sorted by ascending prediction accuracy along the x axis. The best models using only a small subset of modifications almost reach the prediction accuracy of the full linear model. The top one-modification (r max = 0.72, H3K27ac), two-modifications (r max = 0.74, H3K27ac + H4K20me1) and three-modifications models (r max = 0.75, H3K27ac + H3K4me1 + H4K20me1) are very well correlated to expression. Karlic et al., PNAS 107, 2926 (2010) 25

26 Linear model for expression Bar plot showing the frequency of appearance of different histone PTMs in best scoring three-modifications models (142 models) for CD4+ T-cells. Best scoring models are defined as reaching at least 95% of prediction accuracy of the full linear model. Not all modifications are equally important, possibly because of a high degree of redundancy. Karlic et al., PNAS 107, 2926 (2010) 26

27 Promoter methylation Next, the authors separated the promoters into 2779 LCPs (low CpG-content promoters) and 7089 HCPs (high CpG-content promoters). Promoters with normalized CpG content > 0.4 are classified as HCP and the others as LCP. This was motivated by the fact that the nucleosomes in HCPs are almost always decorated with H3K4me3, whereas nucleosomes in LCPs carry this modification only when they are expressed. H3K4me3 is thought to be a mark of transcription initiation. The authors reasoned that if these promoters are differently marked by histone modifications then the predictive power of histone modifications should also differ between these two groups of promoters. Derive separate linear models for both groups. Karlic et al., PNAS 107, 2926 (2010) 27

28 Linear model for expression Frequency of different histone PTMs in best scoring three-modifications models among 50 HCP models and 40 LCP models. Only the top ten modifications are depicted. (A) H4K20me1 and H3K27ac (and possibly H2BK5ac) are significantly overrepresented among the best scoring models for HCPs (p-values hypergeometric test 9.97e-43, 2.58e-31, and 0.003) (B) H3K4me3 and H3K79me1 are significantly overrepresented in the LCPs (p-values of the hypergeometric test 9.71e-36 and 2.1e-34) different modifications are important for the prediction of expression of genes in these two groups. Karlic et al., PNAS 107, 2926 (2010) 28

29 Linear model for expression Normalized cumulative tag counts in the region of -500 base pairs to 3,000 base pairs surrounding the transcription start site of RefSeq genes in CD4+ T-cells for the 5 important modifications identified by our analysis. H3K4me3, H3K27ac, and H2BK5ac have the highest levels at the promoter, with the highest peaks around 100 base pairs downstream of the TSS. H3K79me1 is enriched along the gene body, and H4K20me1 shows two distinct patterns: a peak close to the promoter at a similar position to H3K4me3 and H3K27ac, and a further enrichment across the gene body region. Karlic et al., PNAS 107, 2926 (2010) 29

30 Test whether model is transferable to other cell types Apply trained CD4+ model to CD36+ and CD133+ cells. The gene expression profiles of CD36+ and CD133+ cells are highly correlated to CD4+ T-cells (r = 0.79 and r = 0.82, respectively). Thus, he prediction was restricted to genes with a fold change higher than five. They found high correlation of predicted and measured expression values for both CD36+ (r = 0.75) and CD133+ (r = 0.63) cells. This suggests that the relationship between histone modifications and gene expression is general and not dependent on the cellular context. Karlic et al., PNAS 107, 2926 (2010) 30

31 Roadmap: Integrative analysis of 111 epigenomes How does the epigenomic landscape contribute to cellular circuitry, lineage specification, and the onset and progression of human disease? Roadmap Epigenomics Consortium Nature 518, 317 (2015). 31

32 Mapped modifications H3K4me3 - associated with promoter regions H3K4me1 - associated with enhancer regions H3K36me3 - associated with transcribed regions H3K27me3 - associated with Polycomb repression H3K9me3 - associated with heterochromatin regions H3K27ac and H3K9ac, associated with increased activation of enhancer and promoter regions DNase hypersensitivity denoting regions of accessible chromatin commonly associated with regulator binding DNA methylation, typically associated with repressed regulatory regions or active gene transcripts Roadmap Epigenomics Consortium Nature 518, 317 (2015). 32

33 Data sets available for 111 epigenomes Roadmap Epigenomics Consortium Nature 518, 317 (2015). 33

34 Integrative analysis of 111 epigenomes Chromatin state annotations across 127 reference epigenomes (rows) in a ~3.5-Mb region on chromosome 9. Promoters are primarily constitutive (i.e. unchanged) (red vertical lines), while enhancers are highly dynamic (dispersed yellow regions). Roadmap Epigenomics Consortium Nature 518, 317 (2015). 34

35 Signal tracks for IMR90 Signal tracks for IMR90 (fetal lung fibroblast) showing RNA-seq, a total of 28 histone modification marks, whole-genome bisulfite DNA methylation, DNA accessibility, digital genomic footprints (DGF), input DNA and chromatin conformation information.. Roadmap Epigenomics Consortium Nature 518, 317 (2015). 35

36 Training of recurring 15-states chromatin model Roadmap Epigenomics Consortium Nature 518, 317 (2015). 36

37 Consisteny of chromatin states across genomic positions H3K4me1-associated states (including TxFlnk, EnhG, EnhBiv and Enh) are the most tissue specific, with 90% of instances present in at most 5 10 epigenomes, followed by bivalent promoters (TssBiv) and repressed states (ReprPC, Het). In contrast, active promoters (TssA) and transcribed states (Tx, TxWk) were highly constitutive, with 90% of regions marked in as many as epigenomes. Roadmap Epigenomics Consortium Nature 518, 317 (2015). Quiescent regions were the most constitutive, with 90% consistently marked in most of the 127 epigenomes. 37

38 Relative switching between states More frequent switching found between active states and repressed states. This is consistent with activation and repression of regulatory regions. Roadmap Epigenomics Consortium Nature 518, 317 (2015). 38

39 Summary Combinations of histone modification marks are highly informative of the methylation and accessibility levels of different genomic regions, while the converse is not always true. Genomic regions vary greatly in their association with active marks. Approximately 5% of each epigenome is marked by enhancer or promoter signatures on average, which show increased association with expressed genes, and increased evolutionary conservation. Two-thirds of each reference epigenome on average are quiescent, and enriched in gene-poor and stably repressed regions. Even though promoter and transcription associated marks are less dynamic than enhancer marks, each mark recovers biologically meaningful cell-type groupings when evaluated in relevant chromatin states. Roadmap Epigenomics Consortium Nature 518, 317 (2015). 39